Branched-chain amino acid supplementation during bed rest: effect on recovery.

Bed rest is associated with a loss of protein from the weight-bearing muscle. The objectives of this study are to determine whether increasing dietary branched-chain amino acids (BCAAs) during bed rest improves the anabolic response after bed rest. The study consisted of a 1-day ambulatory period, 14 days of bed rest, and a 4-day recovery period. During bed rest, dietary intake was supplemented with either 30 mmol/day each of glycine, serine, and alanine (group 1) or with 30 mmol/day each of the three BCAAs (group 2). Whole body protein synthesis was determined with U-(15)N-labeled amino acids, muscle, and selected plasma protein synthesis with l-[(2)H(5)]phenylalanine. Total glucose production and gluconeogenesis from alanine were determined with l-[U-(13)C(3)]alanine and [6,6-(2)H(2)]glucose. During bed rest, nitrogen (N) retention was greater with BCAA feeding (56 +/- 6 vs. 26 +/- 12 mg N. kg(-1). day(-1), P < 0.05). There was no effect of BCAA supplementation on either whole body, muscle, or plasma protein synthesis or the rate of 3-MeH excretion. Muscle tissue free amino acid concentrations were increased during bed rest with BCAA (0.214 +/- 0.066 vs. 0.088 +/- 0.12 nmol/mg protein, P < 0.05). Total glucose production and gluconeogenesis from alanine were unchanged with bed rest but were significantly reduced (P < 0.05) with the BCAA group in the recovery phase. In conclusion, the improved N retention during bed rest is due, at least in part, to accretion of amino acids in the tissue free amino acid pools. The amount accreted is not enough to impact protein kinetics in the recovery phase but does improve N retention by providing additional essential amino acids in the early recovery phase.     

Mechanisms responsible for regulation of branched-chain amino acid catabolism

The branched-chain amino acids (BCAAs) are essential amino acids and therefore must be continuously available for protein synthesis. However, BCAAs are toxic at high concentrations as evidenced by maple syrup urine disease (MSUD), which explains why animals have such an efficient oxidative mechanism for their disposal. Nevertheless, it is clear that leucine is special among the BCAAs. Leucine promotes global protein synthesis by signaling an increase in translation, promotes insulin release, and inhibits autophagic protein degradation. However, leucine's effects are self-limiting because leucine promotes its own disposal by an oxidative pathway, thereby terminating its positive effects on body protein accretion. A strong case can therefore be made that the proper leucine concentration in the various compartments of the body is critically important for maintaining body protein levels beyond simply the need of this essential amino acid for protein synthesis. The goal of the work of this laboratory is to establish the importance of regulation of the branched chain alpha-ketoacid dehydrogenase complex (BCKDC) to growth and maintenance of body protein. We hypothesize that proper regulation of the activity state of BCKDC by way of its kinase (BDK) and its phosphatase (BDP) is critically important for body growth, tissue repair, and maintenance of body protein. We believe that growth and protection of body protein during illness and stress will be improved by therapeutic control of BCKDC activity. We also believe that it is possible that the negative effects of some drugs (PPAR alpha ligands) and dietary supplements (medium chain fatty acids) on growth and body protein maintenance can be countered by therapeutic control of BCDKC activity.     

Differential effects of long-term leucine infusion on tissue protein synthesis in neonatal pigs

Leucine is unique among the amino acids in its ability to promote protein synthesis by activating translation initiation via the mammalian target of rapamycin (mTOR) pathway. Previously, we showed that leucine infusion acutely stimulates protein synthesis in fast-twitch glycolytic muscle of neonatal pigs but this response cannot be maintained unless the leucine-induced fall in amino acids is prevented. To determine whether leucine can stimulate protein synthesis in muscles of different fiber types and in visceral tissues of the neonate in the long-term if baseline amino acid concentrations are maintained, overnight fasted neonatal pigs were infused for 24 h with saline, leucine (400 μmol kg⁻¹ h⁻¹), or leucine with replacement amino acids to prevent the leucine-induced hypoaminoacidemia. Changes in the fractional rate of protein synthesis and activation of mTOR, as determined by eukaryotic initiation factor 4E binding protein (4E-BP1) and S6 kinase 1 (S6K1) phosphorylation, in the gastrocnemius and masseter muscles, heart, liver, jejunum, kidney, and pancreas were measured. Leucine increased mTOR activation in the gastrocnemius and masseter muscles, liver, and pancreas, in both the absence and presence of amino acid replacement. However, protein synthesis in these tissues was increased only when amino acids were infused to maintain baseline levels. There were no changes in mTOR signaling or protein synthesis in the other tissues we examined. Thus, long-term infusion of leucine stimulates mTOR signaling in skeletal muscle and some visceral tissues but the leucine-induced stimulation of protein synthesis in these tissues requires sustained amino acid availability.     

Tryptophan Overload in the Pregnant Rat: Effect on Brain Amino Acid Levels and In Vitro Protein Synthesis

The concentration of most amino acids was higher in the brains of 19- and 21-day rat fetuses than in their respective mothers. After an intraperitoneal load of tryptophan to the mother, the intracerebral concentration of several amino acids (including leucine) decreased not only in the mothers, but also in their fetuses. The in vitro incorporation of pHJleucine into proteins in brain postmitochondrial supernatant fractions was enhanced in both the mothers and fetuses after tryptophan administration, but this effect disappeared when protein synthesis was calculated by using specific activities corrected for the amount of unlabeled leucine in the preparation. By this criterion, protein synthesis activity appeared similar in the brains of 19- and 21-day pregnant rats but was higher in their fetuses, especially in the 21-day subjects. Thus, protein synthesis in the brain was not altered by marked changes in the amino acid pool and more profound and prolonged metabolic disturbances must occur to cause permanent damage in the developing brain.     

Metabolism of branched-chain amino acids in starved rats: the role of hepatic tissue

Parameters of branched-chain amino acids (BCAA; leucine, isoleucine and valine) and protein metabolism were evaluated using L-[1-(14)C]leucine and alpha-keto[1-(14)C]isocaproate (KIC) in the whole body and in isolated perfused liver (IPL) of rats fed ad libitum or starved for 3 days. Starvation caused a significant increase in plasma BCAA levels and a decrease in leucine appearance from proteolysis, leucine incorporation into body proteins, leucine oxidation, leucine-oxidized fraction, and leucine clearance. Protein synthesis decreased significantly in skeletal muscle and the liver. There were no significant differences in leucine and KIC oxidation by IPL. In starved animals, a significant increase in net release of BCAA and tyrosine by IPL was observed, while the effect on other amino acids was non-significant. We conclude that the protein-sparing phase of uncomplicated starvation is associated with decreased whole-body proteolysis, protein synthesis, branched-chain amino acid (BCAA) oxidation, and BCAA clearance. The increase in plasma BCAA levels in starved animals results in part from decreased BCAA catabolism, particularly in heart and skeletal muscles, and from a net release of BCAA by the hepatic tissue.     

Enteral glutamine stimulates protein synthesis and decreases ubiquitin mRNA level in human gut mucosa

Effects of glutamine on whole body and intestinal protein synthesis and on intestinal proteolysis were assessed in humans. Two groups of healthy volunteers received in a random order enteral glutamine (0.8 mmol.kg body wt(-1)x h(-1)) compared either to saline or isonitrogenous amino acids. Intravenous [2H5]phenylalanine and [13C]leucine were simultaneously infused. After gas chromatography-mass spectrometry analysis, whole body protein turnover was estimated from traced plasma amino acid fluxes and the fractional synthesis rate (FSR) of gut mucosal protein was calculated from protein and intracellular phenylalanine and leucine enrichments in duodenal biopsies. mRNA levels for ubiquitin, cathepsin D, and m-calpain were analyzed in biopsies by RT-PCR. Glutamine significantly increased mucosal protein FSR compared with saline. Glutamine and amino acids had similar effects on FSR. The mRNA level for ubiquitin was significantly decreased after glutamine infusion compared with saline and amino acids, whereas cathepsin D and m-calpain mRNA levels were not affected. Enteral glutamine stimulates mucosal protein synthesis and may attenuate ubiquitin-dependent proteolysis and thus improve protein balance in human gut.     

Multivalent Induction of Biodegradative Threonine Deaminase

To determine the inducer(s) of the biodegradative threonine deaminase in Escherichia coli, the effects of various amino acids on the synthesis of this enzyme were investigated. The complex medium used hitherto for the enzyme induction can be completely replaced by a synthetic medium composed of 18 natural amino acids. In this synthetic medium, the omission of each of the seven amino acids threonine, serine, aspartic acid, methionine, valine, leucine, and arginine resulted in the greatest loss of enzyme formation. These seven amino acids did not significantly influence the uptake of other amino acids into the cells. Furthermore, they did not stimulate the conversion of inactive enzyme into an active form, since they did not affect the enzyme level in cells in which protein synthesis was inhibited by chloramphenicol. Threonine, serine, aspartic acid, and methionine failed to stimulate enzyme production in cells in which messenger ribonucleic acid synthesis was arrested by rifampin, whereas valine, leucine, and arginine stimulated enzyme synthesis under the same conditions. Therefore, the first four amino acids appear to act as inducers of the biodegradative threonine deaminase in E. coli and the last three amino acids appear to be amplifiers of enzyme production. The term "multivalent induction" has been proposed for this type of induction, i.e., enzyme induction only by the simultaneous presence of several amino acids.     

Isotopic determination of amino acid-urea interactions in exercise in humans

We recently reported that in light exercise (30% VO2max) the oxidation of [1-13C]leucine was significantly increased but the rate of urea production was unchanged (J. Appl. Physiol: Respirat. Environ. Exercise Physiol. 52: 458-466, 1982). We have therefore tested three possible explanations for this apparent incongruity. 1) We infused NaH13CO3 throughout rest and exercise and found that, although altered bicarbonate kinetics in exercise resulted in greater recovery of 13CO2, the difference between rest and recovery was small compared with the increase in the rate of 13CO2 excretion during exercise when [1-13C]leucine was infused. 2) We infused [15N]leucine and isolated plasma urea N to determine directly the rate of incorporation of the 15N. During exercise there was no increase in the rate of 15N incorporation. Simultaneously, we infused [2,3-13C]alanine and quantified the rate of incorporation of 15N in alanine. We found that [15N]alanine production from [15N] leucine more than doubled in exercise, and by deduction, alanine production from other amino acids also doubled. 3) We tested our previous assumption that [1-13C]leucine metabolism in exercise was representative of the metabolism of other essential amino acids by infusing [1-13C] and [alpha-15N]lysine throughout rest and exercise. We found that the rate of breakdown of lysine during exercise was not increased in a manner comparable to that of leucine. Thus, these data confirm our original findings that leucine decarboxylation is enhanced in light exercise but urea production is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems.

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Amino acid availability and age affect the leucine stimulation of protein synthesis and eIF4F formation in muscle

We have previously shown that a physiological increase in plasma leucine for 60 and 120 min increases translation initiation factor activation in muscle of neonatal pigs. Although muscle protein synthesis is increased by leucine at 60 min, it is not maintained at 120 min, perhaps because of the decrease in plasma amino acids (AA). In the present study, 7- and 26-day-old pigs were fasted overnight and infused with leucine (0 or 400 micromol.kg(-1).h(-1)) for 120 min to raise leucine within the postprandial range. The leucine was infused in the presence or absence of a replacement AA mixture (without leucine) to maintain baseline plasma AA levels. AA administration prevented the leucine-induced reduction in plasma AA in both age groups. At 7 days, leucine infusion alone increased eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) phosphorylation, decreased inactive 4E-BP1.eIF4E complex abundance, and increased active eIF4G.eIF4E complex formation in skeletal muscle; leucine infusion with replacement AA also stimulated these, as well as 70-kDa ribosomal protein S6 kinase, ribosomal protein S6, and eIF4G phosphorylation. At 26 days, leucine infusion alone increased 4E-BP1 phosphorylation and decreased the inactive 4E-BP1.eIF4E complex only; leucine with AA also stimulated these, as well as 70-kDa ribosomal protein S6 kinase and ribosomal protein S6 phosphorylation. Muscle protein synthesis was increased in 7-day-old (+60%) and 26-day-old (+40%) pigs infused with leucine and replacement AA but not with leucine alone. Thus the ability of leucine to stimulate eIF4F formation and protein synthesis in skeletal muscle is dependent on AA availability and age.     

Whole body leucine flux in HIV-infected patients treated with or without protease inhibitors

The present study was carried out to assess the effects of protease inhibitor (PI) therapy on basal whole body protein metabolism and its response to acute amino acid-glucose infusion in 14 human immunodeficiency virus (HIV)-infected patients. Patients treated with PIs (PI+, 7 patients) or without PIs (PI-, 7 patients) were studied after an overnight fast during a 180-min basal period followed by a 140-min period of amino acid-glucose infusion. Protein metabolism was investigated by a primed constant infusion of l-[1-(13)C]leucine. Dual-energy X-ray absorptiometry for determination of fat-free mass (FFM) and body fat mass measured body composition. In the postabsorptive state, whole body leucine balance was 2.5 times (P < 0.05) less negative in the PI+ than in the PI- group. In HIV-infected patients treated with PIs, the oxidative leucine disposal during an acute amino acid-glucose infusion was lower (0.58 +/- 0.09 vs. 0.81 +/- 0.07 micromol x kg FFM(-1) x min(-1) using plasma [(13)C]leucine enrichment, P = 0.06; or 0.70 +/- 0.10 vs. 0.99 +/- 0.08 micromol x kg FFM(-1) x min(-1) using plasma [(13)C]ketoisocaproic acid enrichment, P = 0.04 in PI+ and PI- groups, respectively) than in patients treated without PIs. Consequently, whole body nonoxidative leucine disposal (an index of protein synthesis) and leucine balance (0.50 +/- 0.10 vs. 0.18 +/- 0.06 micromol x kg FFM x (-1) x min(-1) in PI+ and PI- groups respectively, P < 0.05) were significantly improved during amino acid-glucose infusion in patients treated with PIs. However, whereas the response of whole body protein anabolism to an amino acid-glucose infusion was increased in HIV-infected patients treated with PIs, any improvement in lean body mass was detected.     

Human soleus and vastus lateralis muscle protein metabolism with an amino acid infusion

The calf muscles, compared with the thigh, are less responsive to resistance exercise in ambulatory and bed-rested individuals, apparently due to muscle-specific differences in protein metabolism. We chose to evaluate the efficacy of using amino acids to elevate protein synthesis in the soleus, because amino acids have been shown to have a potent anabolic effect in the vastus lateralis. Mixed muscle protein synthesis in the soleus and vastus lateralis was measured before and after infusion of mixed amino acids in 10 individuals (28 +/- 1 yr). Phosphorylation of ribosomal protein p70 S6 kinase (p70S6K; Thr389) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1; Thr37/46) was also evaluated at rest and after 3 h of amino acid infusion. Basal protein synthesis was similar (P = 0.126), and amino acids stimulated protein synthesis to a similar extent (P = 0.004) in the vastus lateralis (0.043 +/- 0.011%/h) and soleus (0.032 +/- 0.017%/h). Phosphorylation of p70S6K (P = 0.443) and 4E-BP1 (P = 0.192) was not increased in either muscle; however, the soleus contained more total (P = 0.002) and phosphorylated (P = 0.013) 4E-BP1 than the vastus lateralis. These data support the need for further study of amino acid supplementation as a means to compensate for the reduced effectiveness of calf resistance exercise in ambulatory individuals and those exposed to extended periods of unloading. The greater 4E-BP1 in the soleus suggests that there is a muscle-specific distribution of general translational initiation machinery in human skeletal muscle.     

Physiological rise in plasma leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation factor activation

Protein synthesis in skeletal muscle of adult rats increases in response to oral gavage of supraphysiological doses of leucine. However, the effect on protein synthesis of a physiological rise in plasma leucine has not been investigated in neonates, an anabolic population highly sensitive to amino acids and insulin. Therefore, in the current study, fasted pigs were infused intra-arterially with leucine (0, 200, or 400 micromol.kg(-1).h(-1)), and protein synthesis was measured after 60 or 120 min. Protein synthesis was increased in muscle, but not in liver, at 60 min. At 120 min, however, protein synthesis returned to baseline levels in muscle but was reduced below baseline values in liver. The increase in protein synthesis in muscle was associated with increased plasma leucine of 1.5- to 3-fold and no change in plasma insulin. Leucine infusion for 120 min reduced plasma essential amino acid levels. Phosphorylation of eukaryotic initiation factor (eIF)-4E-binding protein-1 (4E-BP1), ribosomal protein (rp) S6 kinase, and rpS6 was increased, and the amount of eIF4E associated with its repressor 4E-BP1 was reduced after 60 and 120 min of leucine infusion. No change in these biomarkers of mRNA translation was observed in liver. Thus a physiological increase in plasma leucine stimulates protein synthesis in skeletal muscle of neonatal pigs in association with increased eIF4E availability for eIF4F assembly. This response appears to be insulin independent, substrate dependent, and tissue specific. The results suggest that the branched-chain amino acid leucine can act as a nutrient signal to stimulate protein synthesis in skeletal muscle of neonates.     

Effects of branched-chain amino acids on muscles under hyperammonemic conditions

The aim was to determine the effects of enhanced availability of branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) on ammonia detoxification to glutamine (GLN) and protein metabolism in two types of skeletal muscle under hyperammonemic conditions. Isolated soleus (SOL, slow-twitch) and extensor digitorum longus (EDL, fast-twitch) muscles from the left leg of white rats were incubated in a medium with 1 mM ammonia (NH3 group), BCAAs at four times the concentration of the controls (BCAA group) or high levels of both ammonia and BCAA (NH3 + BCAA group). The muscles from the right leg were incubated in basal medium and served as paired controls. L-[1-¹⁴C]leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. We observed decreased protein synthesis and glutamate and α-ketoglutarate (α-KG) levels and increased leucine oxidation, GLN levels, and GLN release into medium in muscles in NH3 group. Increased leucine oxidation, release of branched-chain keto acids and GLN into incubation medium, and protein synthesis in EDL were observed in muscles in the BCAA group. The addition of BCAAs to medium eliminated the adverse effects of ammonia on protein synthesis and adjusted the decrease in α-KG found in the NH3 group. We conclude that (i) high levels of ammonia impair protein synthesis, activate BCAA catabolism, enhance GLN synthesis, and decrease glutamate and α-KG levels and (ii) increased BCAA availability enhances GLN release from muscles and attenuates the adverse effects of ammonia on protein synthesis and decrease in α-KG.     

Leucine and insulin activate p70 S6 kinase through different pathways in human skeletal muscle

Amino acids and insulin have anabolic effects in skeletal muscle, but the mechanisms are poorly understood. To test the hypothesis that leucine and insulin stimulate translation initiation in human skeletal muscle by phosphorylating 70-kDa ribosomal protein S6 kinase (p70(S6k)), we infused healthy adults with leucine alone (n = 6), insulin alone (n = 6), or both leucine and insulin (n = 6) for 2 h. p70(S6k) and protein kinase B (PKB) serine(473) phosphorylation were measured in vastus lateralis muscles. Plasma leucine increased from approximately 116 to 343 micromol/l during the leucine-alone and leucine + insulin infusions. Plasma insulin increased to approximately 400 pmol/l during the insulin-alone and leucine + insulin infusions and was unchanged during the leucine-alone infusion. Phosphorylation of p70(S6k) increased 4-fold in response to leucine alone, 8-fold in response to insulin alone, and 18-fold after the leucine + insulin infusion. Insulin-alone and leucine + insulin infusions increased PKB phosphorylation, but leucine alone had no effect. These results show that physiological concentrations of leucine and insulin activate a key mediator of protein synthesis in human skeletal muscle. They suggest that leucine stimulates protein synthesis through a nutrient signaling mechanism independent of insulin, raising the possibility that administration of branched-chain amino acids may improve protein synthesis in insulin-resistant states.     

NATURE OF THE INHIBITION OF PROTEIN SYNTHESIS BY ETHIONINE AND AMINO ACID UPTAKE IN EARLY SEA URCHIN EMBRYOS

The inhibition of protein synthesis by ethionine reported previously was found to be apparent, and ethionine inhibited only amino acid uptake like other usual amino acids. Even under such strong inhibition of the uptake, the syntheses of protein and DNA remained almost undiminished. The uptake of amino acid mixture by sea urchin embryos in the early cleavage stage was found to be carried out by active transport, since it was temperature-sensitive and was inhibited by 2,4-dinitrophenol. The uptake of an amino acid mixture or of single amino acids, e.g., valine, leucine and phenylalanine, was inhibited nonspecifically by an excess amount of other single amino acids added exogenously. Reflecting the inhibition of amino acid uptake, in vivo incorporation of amino acids into the protein fraction was apparently inhibited by excess amounts of other amino acids. As far as tested, the inhibition seems to be nonspecific and competitive for all amino acid species. The uptakes of leucine and phenylalanine were inhibited mutually by competition, with almost the same Km and Ki.     

Effects of inhibitors of protein degradation on the rate of protein synthesis in Chinese hamster ovary cells

In the absence of serum and amino acids, cultured Chinese Hamster Ovary cells released to the medium two thirds of the leucine produced by protein degradation. Because protein synthesis requires all the amino acids, the loss of leucine implies incomplete reincorporation of the other amino acids as well. Leupeptin (0.45 mg/ml) and chloroquine (up to 40 microM) inhibited protein breakdown by 21 and up to 41%, respectively, and resulted in proportional decreases in protein synthesis. Chloroquine abolished the stimulation of protein breakdown by amino acid deprivation. From the values of protein synthesis and leucine output with and without chloroquine, it is estimated that the stimulation of protein degradation not only permitted continuing protein synthesis but also increased amino acid output. In the presence of serum or amino acids protein breakdown was slower than in their absence and less sensitive to inhibition by chloroquine, but proportional effects on synthesis and degradation were still observed. It is suggested that protein degradation may be necessary for the maintenance of optimum intracellular concentrations of amino acids even in the presence of extracellular amino acids.